Plants like chili peppers and wasabi elicit pungent, almost painful flavors with capsaicin and mustard oil, which have given researchers powerful tools for probing pain-sensing pathways. For other painful products, David Julius and colleagues at the University of California, San Francisco, looked to venomous snakes. In the November 17 issue of Nature, the researchers report the isolation of a protein toxin from the venom of the Texas coral snake that unleashes intense, burning pain on bite victims. Julius and coworkers show that the toxin, MitTx, activates acid-sensing ion channels (ASICs) in sensory neurons, targeting most potently the ASIC1 subtype, a group not well known for roles in nociception.

The findings suggest that ASICs, part of the degenerin/epithelial sodium channel (DEG/ENaC) family of ion channels, play an underappreciated part in detecting painful stimuli. And, just as natural products like capsaicin and mustard oil are useful as probes of specific transient receptor potential (TRP) channels, MitTx now joins the experimental toolbox as a formidable agonist of ASIC1.

Prior research implicated proton-activated ASICs in cardiac and muscle pain from tissue acidosis (reviewed in Deval et al., 2010). In peripheral neurons, the focus has been on ASIC3, because that subtype is selectively expressed in sensory nerve fibers, whereas ASIC1 expression extends through the brain and non-neural tissues. But the MitTx toxin shows that “when you activate ASIC1, it generates a robust pain signal,” Julius told PRF. “So I think it says that we should pay more attention to that subtype.”

Co-first authors Christopher Bohlen and Alexander Chesler discovered MitTx in an unbiased screen for snake venoms that could activate sensory neurons in vitro, using calcium imaging as a readout. Venom from one species with a particularly painful bite, the Texas coral snake (Micrurus tener tener), activated rat trigeminal ganglia neurons, but not sympathetic neurons. Fractionation of the venom revealed that the active component, dubbed MitTx, is a two-protein complex between Kunitz- and phospholipase A2 (PLA2)-like proteins.

To sniff out MitTx’s molecular mechanism, the team studied its effects on cultured trigeminal neurons. Whole-cell patch-clamp experiments showed that the purified toxin evoked currents with a linear current-voltage relationship and that were selective for sodium ions, properties that suggested ASIC involvement. Indeed, in Xenopus oocytes expressing ASICs, MitTx evoked strong, long-lasting currents, even at neutral pH. The response to MitTx was robust for ASIC1a and 1b, exceeding the response to acidic pH. ASIC3 and ASIC2a showed weak responses, and ASIC2b and 4 showed none. Additional electrophysiological experiments supported the idea that MitTx specifically activates the ASIC1s.

In people, the bite of a coral snake produces profound pain, and injection of the purified toxin into the paws of mice recapitulated that response. Consistent with the specificity of MitTx for ASIC1, pain-related behaviors (licking the injected paw) were substantially reduced in ASIC1-deficient mice.

To evaluate which sensory neurons mediate MitTx-triggered pain, the investigators performed calcium imaging in cultured dorsal root ganglion (DRG) neurons. Both nociceptive and non-nociceptive DRG neurons responded to the toxin. But in mice, a spinal capsaicin injection that ablated the subpopulation of TRPV1-expressing nociceptors prevented the animals from showing MitTx-induced pain. That suggests that, while ASIC1s are not confined to nociceptive sensory neurons, nociceptors expressing TRPV1 are the only cells in which ASIC1 activation generates pain. Julius said the experiments, performed in collaboration with UCSF colleague Allan Basbaum (a PRF science advisor), lend fresh support to the idea that TRPV1 expression is a telltale marker of sensory neurons dedicated to pain-sensing functions (see PRF related news story).

Fortunately, most people never experience the bite of the coral snake, but could there be other, endogenous molecules that turn on ASIC1 to potentiate pain? Julius is intrigued that for one ASIC subtype, ASIC2a, MitTx had a weak effect on its own, but greatly enhanced the ability of acidic pH to activate the channel. That result suggests that ASICs might integrate coincident signals from protons and other activators. Small neuropeptides also modulate ASIC activity (Askwith et al., 2000), but seeing potent activation from the much larger MitTx protein toxin “widens your gaze as to the types of things that could interact with this channel,” Julius said. “It says there’s a way that a big protein can snuggle up next to the receptor and activate it in a very persistent and robust way.”

The results also point to antagonists of ASIC1 (whose structure has been determined by X-ray crystallography; Jasti et al., 2007) as potential painkillers. The channels’ widespread expression in the brain and other tissues might be a drawback, Julius noted, but in the meantime, there is a lot to learn about how the channels contribute to pain.